1. Field of the Invention
The present disclosure relates to a method for diagnosing cancer by isolating and enriching glycoproteins which are aberrantly glycosylated due to cancer by using lectin, selecting polypeptides, and quantitatively analyzing the polypeptides.
2. Description of the Related Art
Protein glycosylation is one of the most representative post-translational modifications. When glycoproteins which are abundantly present on the surface of cell membranes receive a command of a specific signal, such as oncogenes, glycosylation occurs aberrantly. Many diseases have been known to correlate with aberrant actions of glycosyltransferases and glycolytic enzymes, which are caused by aberrant signal transduction of oncogenes.
Patterns of the aberrant glycosylation in cancer cells are very diverse, including increase in the size and the number of branches of N-linked glycan, increase in the sialylation and fucosylation, and changes in the glycan size, such as the formation of polylactosamine. With these phenomena proceeded in glycoproteins, these glycoproteins can be used for cancer markers for determining the existence and progression of cancer. Particularly, fucosylation which increases aberrantly in cancer cells, etc. provides the possibility to differentiate between proteins present in cancer cells and those in normal cells, and thus, glycoproteins which are glycosylated aberrantly can be developed as cancer markers for diagnosing cancer (Khien V V, Mao H V, Chinh T T, Ha P T, Bang M H, Lac B V, Hop T V, Tuan N A, Don L V, Taketa K, Satomura S. Clinical evaluation of lentil lectin-reactive alpha-fetoprotein-L3 in histology-proven hepatocellular carcinoma. Int J Biol Markers 2001; 16: 105-111). After termination of required roles, glycoproteins which contain information on cancer are secreted into an extracellular media, or shed from the cellular membrane and released to a media, and therefore, culture media of various cancer cells, lysis products of cancer tissue, and particularly, blood of a patient can be suitable materials for detecting glycoproteins which contain information on cancer, that is, cancer markers.
In protein samples obtained from a normal group and a patient group, the differences in protein glycosylation can be an important cue for differentiating the patient group from the normal group, and therefore, many analytical methods to distinguish these differences have been developed. There are several methods which use the selectivity of lectin for the glycan structure of a glycoprotein to isolate and enrich glycoproteins or glycopeptides only, in order to distinguish the differences in glycosylation. ConA(Concanavalin A), WGA(Wheat germ agglutinin), Jacalin, SNA(Sambucus nigra agglutinin), AAL(Aleuria aurantia lectin), L-PHA(Phytohemagglutinin-L), PNA(Peanut agglutinin), LCA(Lens culimaris agglutinin-A), ABA(Agaricus biflorus agglutinin), DBA(Dolichos biflorus agglutinin), DSA(Datura stramonium agglutinin), ECA(Erythrina cristagalli agglutinin), SBA(Soybean agglutinin), SSA(Sambucus sieboldiana agglutinin), UEA(Ulex europaeus agglutinin), VVL(Vicia villosa lectin), BPL(Bauhinia purpurea lectin), or multilectin which uses mixtures of several lectins is used depending on the various glycan structures (Yang, Z. et al., J. Chromatogr, A, 2004, 1053, 79-88., Wang, Y. et al., Glycobiology, 2006, 16, 514-523). Since this method uses the selectivity of lectin for the glycan structures of glycoproteins, there is an advantage that the selective isolation and enrichment of glycoproteins having specific glycan structures is practicable. Particularly, by removing many proteins which do not exhibit the affinity for lectin through the process of isolating glycoproteins which are selective for lectin, the complexity of analysis samples can be significantly reduced. Isolated and enriched glycoproteins can be analyzed qualitatively and quantitatively using various electrochemical methods, spectrochemical methods, and particularly, mass spectrometric methods.
Example of a method which has been mostly used is lectin-blotting method in which glycoprotein is analyzed using the selectivity of lectin for the glycan structure of the glycoprotein. In addition, the method has been generally used with immunoblotting method which shows high selectivity for specific proteins. Therefore, it is necessary to prepare an antibody against antigen glycoprotein and there is a disadvantage that the method cannot be used for proteins of which antibodies cannot be obtained. In addition, this lectin-blotting method which basically uses a gel-separation technology exhibits many limitations in the analysis speed, quantitative reliability, etc. Recently, antibody-lectin sandwich array method has been able to improve the analysis speed and analysis sensitivity significantly compared to the conventional lectin-blotting method (Forrester, S. et. al., Low-volume, high-throughput sandwich immunoassays for profiling plasma proteins in mice: identification of early-stage systemic inflammation in a mouse model of intestinal cancer. Mol Oncol 2007, 1(2): 216-225). However, obtaining reliable antibody is necessary for this sandwich array method and obtaining antibodies for all glycoproteins which are being discovered on a large scale quickly is difficult.
Meanwhile, a mass spectrometric method is being used as an useful analysis method for a very high-speed and high-sensitivity qualitative and quantitative analysis of very complicated proteomic samples. Particularly, multiple reaction monitoring mass spectrometry (MRM MS) method provides a method which allows to quantify polypeptides of relatively small mass generated from proteolysis, quickly and with high reliability, and the method is particularly useful when antibody against the protein of interest cannot be obtained (Kuhn, E. et. al., Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics 2004, 4(4): 1175-1186). MRM method is a high sensitivity quantitative analysis method, which allows the highly selective analysis of target peptides from very complicated samples, wherein the target peptides which are generated by proteolysis etc. of target proteins intended to be analyzed are isolated by one or more liquid chromatography followed by two stages of mass selection (precursor mass selection and fragment ion selection) (Anderson L, et al., Mol. Cell Proteomics. 2006, 5, 573-588).
Clinical specimens such as plasma proteins have more than 50,000 components and the concentration of protein components thereof is very dynamic (1˜1011, pg/ml), and therefore, it is very difficult to detect and analyze quantitatively trace amount of plasma biomarker proteins from clinical specimens where high concentration proteins coexist, using liquid chromatography-mass spectrometry (LC/MS/MS) approach (Anderson N. L. et al., Mol. Cell Proteomics. 2002, 1, 845-867). Therefore, in order to discover plasma biomarkers of disease, removing high concentration proteins, such as albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), transferring, haptoglobin, etc. which account for more than 90% of plasma to minimize complexity of clinical specimen and analyzing the remaining proteins may be preferable. When the concentration of the target marker protein in the clinical specimen is extremely low in spite of the minimization of sample complexity by removing high concentration plasma proteins and high selectivity for the target peptide by LC-MRM analysis, LOD (limit of detection) and LOQ (limit of quantification) for a cancer marker can be improved by enrichment of the marker protein using immunoaffinity, or by enrichment of hydrolyzed marker peptide.
Thus, the present inventors have found the fact that: hydrolyzed marker peptides derived from marker proteins causing cancer-specific glycosylation could be selected by isolating and enriching glycoproteins aberrantly glycosylated due to cancer by using lectin, hydrolyzing the glycoproteins to obtain polypeptides, and analyzing the polypeptides quantitatively; and cancer could be diagnosed by quantitatively analyzing the marker peptides. The prevent inventors completed the present invention based on the fact.